Brain dopamine (DA) signaling is impacted by psychostimulants and is believed to promote addictive behaviors. Termination of DA neurotransmission is achieved primarily by the DA transporter (DAT). In addition to direct inhibition by drugs like cocaine, DAT cell surface expression is rapidly regulated by its inhibitors and substrates and by activation of DA D2R autoreceptors (D2Rs). Cdk5 is a neuronal protein kinase that regulates striatal post-synaptic DA receptor signaling pathways implicated in addiction. Cdk5 can also alter DA neurotransmission via actions at the DA neuron itself. My preliminary data show that pharmacological inhibition of Cdk5 dually regulates rat dorsal striatal (dSTR) DAT velocity, which likely occurs via two distinct mechanisms. Furthermore, my data suggest that Cdk5 is a positive regulator of DAT function. The goals of my proposal are to determine 1) the cellular trafficking mechanisms by which Cdk5 inhibition dually regulates the DAT and 2) if cocaine-induced increase in cell-surface DAT expression occurs via a D2R-dependent phosphorylation of Cdk5 in DA neurons. Aim #1 will characterize further DAT downregulation by determining how Cdk5 inhibition affects cell-surface DAT expression in rat dSTR synaptosomes and in vivo DA clearance in intact rat dSTR via chronoamperometry. To determine if Cdk5 inhibition down-regulates DAT via a Cdk5-mediated, dynamin-dependent mechanism, I will use differentiated PC12 cells stably transfected with human DAT in conjunction with short interfering RNA (siRNA) technology and the novel fluorescent DAT substrate ASP+ for monitoring real-time DAT-mediated uptake in single cells. Aim #2 will explore the mechanism(s) underlying up-regulation of DAT activity and presumed cell-surface expression, which occurs after Cdk5 inhibitor wash out, in dSTR synaptosomes. I will determine if the mitogen-activated protein kinase (MAPK) signaling pathway is involved. In addition, using primary cultures of post-natal ventral mesencephalic DA neurons, I will use live cell imaging of ASP+ accumulation, immunoblot analysis and cell-surface biotinylation to determine if cocaine rapidly up-regulates cell-surface DAT expression and, thus, its activity via D2R-mediated phosphorylation of Cdk5. Diseases linked to dysfunctional DA neurotransmission (e.g. drug addiction and Parkinson's disease) continue to be a major public health burden with a significant impact on society. The search for therapies that offer more than symptomatic relief remains a top priority. Identification of novel therapeutic targets is contingent upon our understanding of how key molecular players that govern DA neurotransmission (i.e. DAT) are regulated.